How to reduce and prevent evaporation crosstalk

Evaporation is a key step in the sample preparation workflow, either to concentrate analyte of interest or to exchange a solvent for one more compatible with the subsequent analytical technique. Sample extracts are typically collected in a 96-well collection plate and for evaporation are processed under a controlled flow of air or nitrogen. Sample cross-contamination or ‘crosstalk’ occurs when analytes are carried out with the volatilized solvents, as depicted in Figure 1.

Fig. 1

This phenomenon is more likely to happen when the concentration of the analyte in the “hot spot” well is considerably higher than in the surrounding wells. Analyte crosstalk can lead to the detection of extraneous signals in adjacent and/or surrounding wells, potentially resulting in false positive results leading to serious consequences in any assay.

Strategies to prevent crosstalk

There are multiple strategies which can be adopted to prevent analyte crosstalk. One of the simplest ways to reduce evaporation crosstalk  is to avoid overfilling the wells. Filling them to 75% or less of their maximum volume can effectively reduce liquid splash and capillary action, especially for liquids with low surface tension and high analyte sample concentration. Additionally, starting the evaporation with low gas flow and maintaining an appropriate gas-liquid distance (1-3 cm) is crucial to reduce cross-contamination.

Volatile analytes, such as amphetamine, can co-evaporate with non-polar solvents and migrate to other wells during evaporation. Adjusting the sample pH produces more polar salts that are less volatile, effectively reducing crosstalk. The downside of this approach is that strong acidic or basic conditions may cause hydrolysis or analyte degradation during evaporation. While valuable, this strategy may not work for all volatile analyte(s), especially those whose volatility is less impacted by pH modification. To prevent or minimize evaporation crosstalk, additional precautions can be taken by using the Biotage® ATC Plate Adapter (Figure 2).

Fig. 2

Solving crosstalk with the Biotage® ACT Plate Adapter

The Biotage® ACT Plate Adapter is a novel solution to hot spot cross-contamination during evaporation. It provides a mechanical barrier against volatile analyte(s) transfer. The adapter fits securely on top of the 96-well collection plate with ‘chimneys’ directing the gas flow into the wells during evaporation. As the solvent evaporates, the vapors are directed away from sample wells, minimizing eddy formation and reducing the open surface area at the top of the wells. This design helps prevent vapor that carries the analytes from mixing with gas and re-entering adjacent wells, thereby reducing the risk of cross-contamination. Biotage® ACT Plate Adapter’s universal design makes it a cost-effective accessory that can significantly decrease the likelihood of falsely elevated results in any 96-well-based assay. Made of anodized aluminum, it is easily cleaned between runs to prevent any potential carryover.

When evaporating samples with concentrated volatile analytes in a 96-well plate, the Biotage® ACT Plate Adapter is the preferred choice to prevent crosstalk contamination. This approach helps avoid the introduction of strong acids or bases into the sample, thereby reducing analyte degradation. Comprehensive information and data comparisons on these three approaches for crosstalk prevention are provided in the TurboVap® 96 Dual White Paper.